The proliferation and differentiation of cells in multicellular organisms is subject to a highly regulated process. A distinguishing feature of cancer cells is the absence of control over this process; proliferation and differentiation become deregulated resulting in uncontrolled growth. Significant research efforts have been directed toward better understanding this difference between normal and tumor cells. One area of research focus is growth factors and, more specifically, autocrine growth stimulation.
Growth factors are polypeptides which carry messages to cells concerning growth, differentiation, migration and gene expression. Typically, growth factors are produced in one cell and act on another cell to stimulate proliferation. However, certain malignant cells, in culture, demonstrate a greater or absolute reliance on an autocrine growth mechanism. Malignant cells which observe this autocrine behavior circumvent the regulation of growth factor production by other cells and are therefore unregulated in their growth.
Study of autocrine growth control advances understanding of cell growth mechanisms and can lead to important advances in the diagnosis and treatment of cancer. Toward this end, a number of growth factors have been studied, including insulin-like growth factors (“IGF1” and “IGF2”), gastrin-releasing peptide (“GRP”), transforming growth factors alpha and beta (“TGF-a” and “TGF-b”), and epidermal growth factor (“EGF”).
The present invention is directed to a recently discovered growth factor. This growth factor was first discovered in the culture medium of highly tumorigenic “PC cells,” an insulin-independent variant isolated from the teratoma derived adipogenic cell line 1246. This growth factor is referred to herein as “GP88” or “GP88.” GP88 has been purified and structurally characterized. Amino acid sequencing of GP88 indicates that GP88 has amino acid sequence similarities with the mouse granulin/epithelin precursor.
Granulins/epithelins (“grn/epi”) are 6kDa polypeptides and belong to a novel family of double cysteine rich polypeptides. U.S. Pat. No. 5,416,192 (Shoyab et al.) is directed to 6 kDa epithelins, particularly epithelin 1 and epithelin 2. According to Shoyab, both epithelins are encoded by a common 63.5 kDa precursor, which is processed into smaller forms as soon as it is synthesized, so that the only natural products found in biological samples are the 6 kDa forms. Shoyab et al. teaches that the epithelin precursor is biologically inactive.
Contrary to the teachings of Shoyab et al., the inventor's laboratory has demonstrated that the precursor is not always processed as soon as it is synthesized. Studies, conducted in part by this inventor, have demonstrated that the precursor (i.e., GP88) is in fact secreted as an 88kDa glycoprotein with an N-linked carbohydrate moiety of 20 kDa. Analysis of the N-terminal sequence of GP88 indicates that GP88 starts at amino acid 17 of the grn/epi precursor, demonstrating that the first 17 amino acids from the protein sequence deduced from the precursor cDNA correspond to a signal peptide compatible with targeting for membrane localization or for secretion. Also in contrast to the teachings of Shoyab et al., GP88 is biologically active and has growth promoting activity, particularly as an autocrine growth factor for the producer cells.
Multi-cellular organisms require a careful balance between the production and destruction of cells in tissues throughout the body. Apoptosis, or programmed cell death, is a controlled process by which damaged cells or cells replicating outside of normal cellular control can be eliminated without causing the tissue destruction and inflammatory responses often associated with acute injury and necrosis.
Recent studies indicate that apoptosis is controlled through a metabolic pathway which may be induced by a variety of signals (e.g., hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing radiation, and viral infection). See, e.g., U.S. Pat. Nos. 6,586,395 and 6,570,002. The Bcl-2 family of genes regulate apoptosis in many cell types. The normal function of Bcl-2 is to block apoptosis in response to a variety of signals (e.g., radiation, hyperthermia, growth factor withdrawal, glucocorticoids, and multiple classes of chemotherapeutic agents). Id. Thus, blocking the activity of Bcl-2 induces apoptosis. Zhang et al., Clinical Cancer Research, 5:2971-2977 (October 1999). For example, the anti-estrogen compound tamoxifen down regulates Bcl-2 and thus induces apoptosis in breast cancer cells. Id. In addition to inhibiting the growth promoting effect of estrogen, tamoxifen has also been shown to induce programmed cell death in breast cancer cell lines and in clinical samples. Failure to undergo apoptosis in response to tamoxifen confers tamoxifen resistance.
Anti-estrogen therapy is widely used for the treatment of breast cancer. Tamoxifen has been the major agent used for this purpose. The activity of tamoxifen is typically observed in breast tumors that are estrogen receptor positive, since estrogen is the major growth stimulator for these types of tumors. However, after prolonged anti-hormonal therapy, breast cancer can progress from an estrogen sensitive to insensitive state. Breast tumors that were previously growth inhibited by tamoxifen and other anti-estrogen compounds then become resistant to anti-estrogen treatment.
The morphological changes induced by tamoxifen are characteristic of the changes induced by apoptosis. Up-regulation of Bcl-2 by HER2 suppresses tamoxifen-induced apoptosis in breast cancer cells. Kumar et al., Clin. Cancer Res. 1996 Jul.2(7):1215-9. Thus, modulation of Bcl-2 levels provides a mechanism for inducing apoptosis. Inducing apoptosis leads to tumor regression by eliminating, shrinking, and destroying tumor cells. Trauth et al., Science. 1989 Jul. 21;245(4915):301-5. Administration of antisense oligonucleotides directed to the anti-apoptotic Bcl-2 gene induces tumor regression in mice in vivo. Elez et al., Oncogene (2003) 22: 69-80.
What is needed are new methods and compositions for inducing apoptosis and restoring sensitivity to the antitumorigenic effects of antiestrogen therapy and cytotoxic therapy.